Digestive disorders play an increasingly greater role in the general medical and internal medical practice. Such digestive disorders are in many cases the consequence of a more or less pronounced deficiency in so-called pancreatic enzymes. In a healthy state, these enzymes are synthesized in the pancreas by highly specialized cells, the so-called acinic cells, and secreted by exocytosis through juice glands and the main pancreatic duct into the duodenum. The daily amount of pancreatic secretion is about 2 liters. In addition to fat digesting lipase, the pancreatic secretion also contains enzymes for the digestion of proteins (trypsin, chymotrypsin and carboxypeptidases) and carbohydrates (α-amylase). The secretion of pancreatic enzymes is exactly controlled by endogenous control mechanisms by means of hormones, such as gastrin, secretin and pancreozymin. This control system can be disturbed by a large number of causes to result in a reduction of pancreatic enzyme secretion or in a complete subsiding of the exocrine pancreatic function. This in turn causes that the chyme is not digested in the small intestine, and a digestive disorder occurs. This disease of the digestive tract, which is also referred to as exocrine pancreatic insufficiency (EPI), can have different causes. In addition to dyspepsia caused by medicaments, chronic atrophic gastritis and chronic pancreatitis, frequently caused by alcohol consumption, disorders caused by surgery (e.g., Billroth I and II, vagotomy, pancreas resection) and cystic fibrosis are etiologic factors of pancreatic insufficiency. At any rate, chronic digestive disorders are of considerable social-medical and thus economic importance, because the symptoms frequently cause the patients to be nondescript and have a shortened expectation of life.
Pancreatogenic digestive disorders and especially EPI cause a lot of complaints in the patients, such as diarrhea, mass stools, sensations of repletion, upper abdominal complaints, weight loss etc.
Irrespective of the causes and the manifestation of pancreatogenic digestive disorders or EPI to avoid malnutrition related morbidity and mortality, it is pivotal to commence a substitution therapy with enzymes as soon as EPI is diagnosed. This means that the lacking enzymes, predominantly lipase, protease and amylase, but also other enzymes, must be supplied externally. In the therapy, the enzymes are taken in orally by the patient mostly in the middle of the meal and go through the stomach and arrive in the small intestine, where they perform digestion of the chyme and thus adopt the function of the lacking endogenous pancreatic enzymes. The preparations employed must contain a sufficient amount of enzymes. In addition, the enzymes must be provided in an enteric formulation, have a small particle size and be completely bioavailable in the digestive tract.
For treating digestive disorders based on the lacking of pancreatic enzymes often pancreatic enzyme replacement therapy (PERT) based on the substitution/replacement of the leading enzyme lipase and the protease, is used. For PERT a wide variety of enzyme preparations are already on the market. These are partly based on pancreatic enzymes from pigs, such as the preparations Combizym®, Festal®, Pankreon®, Kreon®, Panzytrat®, Meteozym® or Enzym-Lefax N® Preparations containing pancreatic enzymes, so called pancreatic enzyme products or PEPs, are mostly obtained from pigs from slaughter, for example, pancreas, of pigs. The final product of the preparation process is pancreatin. PEPs are composed of porcine lipase, amylase, and protease and are used in patients with EPI secondary to cystic fibrosis, chronic pancreatitis, and pancreatectomy.
In 1938, PEPs were exempted from the Food, Drug, and Cosmetic Act of 1938 and never underwent a formal Food and Drug Administration (FDA) review process (Giuliano C A L Dehoome-Smith M L, Kale-Pradhan P B (2011) Pancreatic enzyme products: digesting the changes. Ann Pharmacother. 45(5):658-66.
PEPs from pig origin cannot be employed with patients suffering from digestive disorders who have a pig protein allergy. In addition, pigs are considered a natural reservoir of human-pathogenic influenza viruses and a vast number of viruses from porcine origin, so that contamination of pancreatin with such viruses cannot be ruled out. In other words, pancreatic tissue, which would present slaughter waste, if not further processed, can exhibit a high degree of viral contamination. In consequence based on its natural origin, the pancreatic tissue, pancreatin and PEP also can be contaminated with viruses from porcine origin. It has to be emphasized that the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) sets a very high standard in its guideline ICH Topic Q 5 A (R1) and demands as the best reasonable assurance that the product is free of virus contamination. The Center for Drug Evaluation and Research (CDER) of the US FDA already requested intensive risk mitigation strategies for lipase containing PEPs like Creon.
This, because there is a risk for contamination of PEPs with Porcine Parvovirus and Porcine Circovirus as well as significant number of swine viruses that are known human pathogens.
Unfortunately, neither manufacturer thus far has found any viral inactivation method that can successfully demonstrate acceptable virus clearances of PEP without also degrading or reducing the pancreatic enzymes, particularly lipase, to unacceptable levels. Due to further limitations associated with analytical testing of such a complex biological material, like pancreatin, it will be difficult to determine what degradants may be introduced into the product as a result of any added viral clearance steps. In conclusion, process steps that can be effective against viruses have a high potential for changing the nature of PEPs, thus having a potentially serious impact on the drug's quality, safety and efficacy. However, because there are almost no alternative resources on the market for lipases than PEPs, they still represent the only permitted drug compositions for the treatment of EPI.
In addition, for particular groups of patients, a disadvantage of the use of PEPs is the origin from pig. Usually, the pancreas of pigs is used, which cannot be tolerated by patients of Judaic or Islamic religion due to religious instructions. Furthermore pancreatin is a homogenate from the cells of pancreatic tissues (usually from pigs). Due to the rupture of a large number of acinic cells, it contains, in addition to pancreatic enzymes, a wide variety of other enzymes and proteins as well as further high and low molecular weight compounds. The composition of pancreatin is due to its industrial preparation process. To obtain pancreatin, pancreas of pigs are deep-frozen as quickly as possible after slaughtering, collected and broken up mechanically. For the stabilization and activation of the enzymes, various additives are added to the homogenate. This is followed by defatting with organic solvents, such as acetone, the removal of fibrous substances, and dewatering and drying by lyophilization. In view of the problems associated with the management of organic solvents and the costs thereof, there thus remains a need for a method of enzyme production which minimises the use of organic solvents or work without of organic solvents. For the preparation of particular dosage forms, further galenic processing may be effected into micropellets, tablets, capsules, pastes, creams, gels, oils or other formulations. Frequently, pancreatin based PEPs are mixed with various support materials and buffer substances. Further, granulated pancreatin is coated with acid-stable films or lacquers for protection against the low pH value of human gastric juice. The two latter processing steps are to ensure that the acid-labile PEPs can fulfill their digestive function at the target site, the duodenum (small intestine), but prevent the enzymes from being active in the acidic upper Duodenum of many pancreatitis Patients.
A newer PEP, which contains lipase, is Zenpep®. Zenpep® is a combination of porcine-derived lipases, proteases and amylases from pigs for slaughter indicated for the treatment of EPI due to cystic fibrosis or other conditions. In opposite to common marketed PEPs for EPI, Zenpep® does not show great variability in the amount of enzymes included in each capsule. The variability of the common PEPs is due in part to the manufacturer practice of overfilling capsules to account for enzyme degradation that occurs over the course of the product's shelf life. Variability in the product's enzyme content can lead to inconsistent therapeutic effects by either providing too much or too little of the required enzymes, which may lead to the suboptimal treatment of the patient's EPI. In addition, overfilled products may increase the risk of fibrosing colonopathy, which has been associated in some reports with long-term exposure to high-dose PERT. These problems can be avoided by the application of Zenpep®. However, Zenpep® contains the same porcine pancreatic lipase like common PEPs. In consequence porcine pancreatic lipase in Zenpep® becomes efficient only in the presence of porcine colipase in the duodenum. Thus, Zenpep® also nearly an unpurified protein mixture from pigs for slaughter, because purification in the manufacturing process would lead to a potential loss of the colipase. Additionally, Zenpep® can also contain viral contamination and, because of the necessary precipitation and defattening steps, residual organic solvents as described for PEPs. In consequence, as for common PEPs for PERT, porcine pancreatic lipase in this composition can be contaminated with viruses and/or organic solvents.
In order to circumvent the use of pig pancreatic tissue as source for lipase and protease, some researchers suggested the use of enzymes from fish or other marine animals generally described in FR No. 1015566, as well as compositions of enzymes from the gastrointestinal tract of krill (crustaceans from the class Euphausiaceae) and capelin fish (U.S. Pat. No. 4,695,457). However these natural resources are difficult to handle and to process for characterized enzyme preparations in industrial scale and in consequence enzyme preparation containing lipases from these origins never reached the market.
Due to the problems of contamination with viruses and organic solvents characterizing conventional enzyme preparation from pig pancreatic tissue, the use of microbially-derived enzymes as alternatives to porcine-derived PEP has been proposed. For example, U.S. Pat. No. 6,051,220 describes compositions comprising one or more acid stable lipases and one or more acid stable amylases, both preferably of fungal origin. United States patent application 2004/0057944 describes compositions comprising Rhizopus delemar lipase, Aspergillus melleus protease and Aspergillus oryzae amylase.
In part, some enzyme preparations therefore also contain microbial enzymes from mold extracts, such as Nortase® and Combizym®. In spite of their resistance to acid and although they do not depend on colipase, their clinical efficacy is low due to rapid intraluminal inactivation by bile salts and proteases.
A recombinant enzyme preparation in development, which contains a bile salt stable lipase, is a drug with the brand name Kiobrina, which is a recombinant human bile-salt-stimulated lipase (rhBSSL). This rhBSSL shall improve the digestion and absorption of essential fatty acids, such as long-chain poly-unsaturated fatty acids but also cholesterol esters in the human gut. rhBSSL is expressed and produced in mammalian cells and is developed by Sobi for enzyme therapy to improve growth and development in preterm infants receiving pasteurized breast milk and/or formula. The rationale for substitution of rhBSSL in pasteurized breast milk or infant formula is to restore the natural lipase activity level that is either lost on pasteurization or totally absent in formula. A disadvantage is, that the enzyme is active only in the presence of primary bile salts, which are often insoluble and, thus, not available in the duodenum, especially under the condition of exocrine pancreatic insufficiency. Furthermore the developing company Sobi announced that clinical data show that the rBSSL did not meet its primary endpoint and, thus, the rhBSSL is does not fulfill its function for enzyme substitution therapy in the human gastrointestinal tract.
Another recombinant lipase for the treatment of exocrine pancreatic insufficiency is the crystallized, purified cross-linked Pseudomonas/Burkholderia cepacia lipase with of molecular weight of 30 kDa, which is part of an enzyme composition named ALTU-135/TheraCLEC®/Trizytek®/Liprotamase®/Sollpura® (now with the brand name Sollpura®) developed by Anthera Pharmaceuticals and which is also disclosed in U.S. Pat. No. 7,718,169.
This microbial lipase is of bacterial origin, which is described in United States patent application 2001/0046493, can be produced by recombinant DNA technology. The enzyme is secreted in the culture medium, and requires a complex production process for purification and crystallization in order to stabilize the enzyme for administration in the human gastro intestinal system. After crystallization, the lipase crystals have to be chemically and covalently cross-linked in order reach sufficient acid stability. In vitro studies suggest that this modification called cross-linked enzyme crystals (CLEC) process increases stability of the lipase and that the cross-linked lipase is insoluble at acidic pH representative of the stomach. However, it has been shown that diffusion effects have been a serious problem for the practical use of crystalline enzymes (Alter, G. M., Leussing, D. L., Neurath, H. Bert L. Vallee, B. L., 1977) Kinetic Properties of carpoxipeptidase B in solution and crystals. Biochemistry 16 (16): 3663-3668). Furthermore it has been shown, that CLEC lipases show a significantly reduced enzyme activity on certain substrates compared to soluble enzymes (Margolin, A. L. (1995) Novel crystalline catalysts. Trends in Biotechnology 14 (7): 223-230).
Moreover, the Pseudomonas/Burkholderia cepacia lipase has a broad pH optimum from pH 4-8 with a strong decline of activity for pH values larger than 8. However, Pseudomonas/Burkholderia cepacia lipase is not active under pH conditions significantly larger than pH 8. In consequence this enzyme cannot support the digestion of lipids under strong alkaline conditions (larger than pH 8.5 or higher). One other problem for Pseudomonas/Burkholderia cepacia lipase is, that the enzyme is a protein from a pathogenic bacterium. Pseudomonas/Burkholderia cepacia (explanation: formerly known as Pseudomonas cepacia, the bacterium now is known as Burkholderia cepacia) is an important human pathogen which most often causes pneumonia in immunocompromised individuals with underlying lung disease such as cystic fibrosis or chronic granulomatous disease.
Patients with cystic fibrosis are at risk for acquiring the well known “Burkholderia cepacia syndrome and in consequence the so called “Burkholderia cepacia syndrome” is a serious condition in patients with cystic fibrosis that does not always respond well to treatment.
In the case of such a Pseudomonas/Burkholderia cepacia infection long-term active memory is acquired following infection by activation of B and T cells, while some of their offspring become long-lived memory cells. Throughout the lifetime of human, these memory cells as part of the “adaptive immune system”, remember each specific pathogen encountered and can mount a strong response if the pathogen is detected again.
Under the conditions of oral treatment with Pseudomonas/Burkholderia cepacia lipase the oral uptake of enzymes from Pseudomonas/Burkholderia cepacia as part of enzyme replacement therapy (in the case of cystic fibrosis) represents an incorporation of exogenous antigens. The antigens will be detected by the adaptive immune system as pathogens and would result 1. in the activation of the immune system (so called immunological memory), 2. the generation of antibodies against the enzymes and 3. the risk of an exaggerated immune response in the intestine mucosa including potential allergic and autoimmune reactions and sepsis.
This risk of an of exaggerated immune response in the case of a medication of cystic fibrosis with Pseudomonas/Burkholderia cepacia enzymes would be counterproductive for the treatment of patients and would exacerbate the patients' health situation.
The combination of insufficient efficacy and the risk of the above described detrimental side effects have already raised serious concern of regulatory agencies. In 2011, the Gastrointestinal Drugs Advisory Committee of the Food and Drug Administration (FDA) of the US Department of Health and Human Services rejected the finding that Sollpura®'s (the enzyme preparation with Pseudomonas/Burkholderia cepacia lipase) benefits outweighed its risks, arguing that additional efficacy data were needed before it could conclude that it worked in patients better than existing porcine derived pancreatic enzyme products.
Finally it has been shown that there is substantial evidence that Sollpur® is less efficacious than the porcine-derived PEPs and appears to expose patients with EPI to greater risk (Carome M. Wolfe S. Testimony to the FDA Gastrointestinal Drug Advisory Committee regarding liprotamase—risk: benefit assessment; ethics of further clinical trials. Washington D.C.: Public Citizen Research Group. Jan. 12, 2011)
Moreover, the U.S. Pat. No. 5,998,189 discloses a recombinant acid stable dog gastric lipase produced in E. coli and claims the expression of acid stable dog gastric lipase in E. coli as well as in other prokaryotic and eukaryotic expression systems. Additionally, the expression and extraction of this acid stable dog gastric lipase in transgenic corn has been demonstrated Zhong, Q., GU, Gu, Z. and Glatz, C. E. (2006) Extraction of recombinant dog gastric lipase from transgenic corn seed. J. Agric. Food Chem. 54: 8086-8092).
However, dog gastric lipase shows a very low pH optimum at pH 4 with a very narrow pH profile from pH 3-pH 5 (Carriere, F., Moreau, H., Raphel, V., Laugier, R., Benicourt, C., Junien, J.-L. and Verger, R., 1991) Purification and biochemical characterization of dog gastric lipase. Eur. J. Biochem. 202: 75-83)
The goal of an enzyme preparation containing lipase displaying the highest efficacy at the lowest dose, and characterized by a well-defined safety profile, remains of great importance to all patients suffering from pancreatic insufficiency, including those in the cystic fibrosis community.
TABLE 1Summary enzyme preparation for PERT approachespHotimumname and brand nameofon marketsourceenzymeslipaseremarkslimitationsPEPs (Creon ®,Pancreatin orpancreatic enzymes from7 to 8no biotechnological process,Cotazyme ®,pancreatic extract drugpigno containment process, soUltrase ®,products from pigcontamination with virusesViokase ® etc.)pancreas tissueand bacteria are possible dueto non-steralized tissue fromslaughter houseZenpep ®Pancreatin orpancreatic enzymes from7 to 8no biotechnological process,pancreatic extract drugpig with defined enzymeno containment process, soproducts from pigactivitycontamination with virusespancreas tissueand bacteria are possible dueto non-steralized tissue fromslaughter houseno producttissue from krill andlypolytic andno pHno biotechnological process,capelin fishproteolytic enzymesoptimiumno containment process, sofrom gastrointestinaldisclosedcontamination with virusestractand bacteria are possible dueto non-steralized tissue withwaste character of the naturalsourceNortase ®/Microbial - mouldRhizopus oryzae/delemar6.6 to 7.5Rhizopus oryzae/delmar is anCombizym ®funguslipase, Aspergillusopportunistic humanmelleus protease andpathogen, potential immuneAspergillus oryzaereaction if previousamylasePseudomonas/Burkholderiacepacia infection, lipaseunstable under physiologicalbile salt concentrationsKiobrina ®recombinant human bile7.3 to 8.6complex and expensivesalt stable lipaserecombinant production(rhBSSL)process in animal cell linesALTU-Microbial - culture ofrecombinant6.5 to 9cross-linkedPseudomonas/Burkholderia135/TheraCLEC ®/E. coli bacteriaPseudomonas/Burkholderiaenzymecepacia opportunistic humanTrizytek ®/cepacia lipasecrystalspathogen, potential immuneLipratamase ®/(CLECreaction if previousSollpura ®process)Pseudomonas/Burkholderiacepacia infection, low activityunter acidic conditionsno productMicrobial - culture ofacid stable dog gastric3 to 5low activity under alkalineE. coli bacterialipase produced in E. coliconditions(http://www.google.com/patents/US5998189)Merispase ®transgenic comacid stable dog gastric3 to 4low activity under alkalinelipase in transgenic cornconditionsno productMicrobial - culture ofacid stable Tetrahymena3.5 to 4.5low activity under alkalineT. thermophilalipasesconditions
In most patients, lipid digestion cannot be completely normalized by current standard therapy. Furthermore the production of lipases for human administration is cumbersome and expensive. The instant invention addresses these issues.